Cellular microarrays for screening differentiation factors

ABSTRACT

Provided is a microarray platform for the culture of cells atop combinatorial matrix mixtures; enabling the study of differentiation in response to a multitude of microenvironments in parallel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application Ser. No. 60/645,556, filed Jan. 20, 2005, thedisclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH

The U.S. Government has certain rights in this invention pursuant toGrant No. DK56966 and DK65152 awarded by the National Institutes ofHealth (NIH).

FIELD OF THE INVENTION

The invention is directed to cell culture techniques and systems andmore particularly to cellular microarrays for screening factors thatmodulate cell differentiation, growth, survival, and/or activity.

BACKGROUND

Microenvironments play a role in modulating cell growth, differentiationand activity. However, current in vitro environments used to culturecells fail to provide optimal microenvironments to simulate in vivocell/tissue growth and differentiation. Cell culture techniques and theunderstanding of the complex interactions cells have with one anotherand the surrounding environment have improved in the past decade. Thereis now a better understanding of the role extracellular matrix materialsplay in the proliferation and development of artificial tissues invitro. Historically cell culture techniques and tissue development failto take into account the necessary microenvironment for cell-cell andcell-matrix communication as well as an adequate diffusional environmentfor delivery of nutrients and removal of waste products.

While many methods and bioreactors have been developed to grow tissuemasses for the purposes of generating artificial tissues fortransplantation or for toxicology studies, these bioreactors do notadequately simulate in vitro the mechanisms by which nutrients and gasesare delivered to tissue cells in vivo.

SUMMARY OF THE INVENTION

The invention provides a cell culture substrate, comprising a pluralityof microspot islands or microwells in an array, wherein the microspotislands or microwells comprise an insoluble factor or an insoluble andsoluble factor, wherein the insoluble factor promotes cellular adhesion.In one aspect, the cell culture substrate comprises a polymerizedbiopolymer such as a hydrogel. In another aspect, the insoluble factoris an extracellular matrix protein. The culture substrate may furthercomprising a plurality of microfluidic channels connecting one or moremicrospot islands or microwells to a fluid flow.

The invention also provides a method of making a microarray. The methodcomprises spotting a plurality of locations on a substrate with anadherence material. In one aspect, the adherence material is anextracellular matrix protein. The substrate may be layered with ahydrogel. The hydrogel may be etched at each of the plurality oflocations to form a microwell.

The culture substrates of the invention are useful for culturing one ormore cell types that adhere to each location comprising an insolubleand/or soluble material (e.g., an adherence material).

The invention also provides a method of making a culture substrate,comprising spotting a material on the substrate using a device capableof spotting from about 1 to about 1000 nanoliters of material togenerate an island of material.

The invention also provides a microarray formed by the methods andprocesses of the invention. A culture system is also provided by theinvention.

The invention provides an assay system useful for protein production,clone amplification and selection, and/or screening of compounds (e.g.soluble and insoluble) useful for cell type maturation, growth anddifferentiation. The assay system comprises contacting a microarray ofthe invention with one or more cell-types and measuring an activityselected from gene expression, cell function, metabolic activity,morphology, and a combination thereof.

Other aspects of the invention will be understood from the drawings, thedetailed description, and examples provided below.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic depicting a microarray of the invention.

FIG. 2A-F is a schematic depiction of fabrication and use of amicroarray of the invention. Integration of wells, sustained release,microfluidics and electronic sensors and actuators can be included inthe methods and systems. (A) Shows the creation of hydrogel wells byspotting periodic acid etch solution on acrylamide gel with DHEBAcross-linker. (B) Shows two examples for potential use: spotting andimmobilizing proteins to bottom of wells, and spotting and gelation ofprepolymer solution containing Extracellular Matrix (ECM) materials andsoluble factors for continuous local release. (C) Illustration ofsustained release mode of use. Hydrogel wells have been filled withdegradable matrix that releases soluble factors locally. (D) Overlayingwith microfluidic channels for mixing soluble factors and delivery toculture chambers. (E) Integration of electronic components, such asheating, digital processing, analog processing, and sensors. (F) Aschematic demonstrating a top and side view of microfluidic delivery andparallel screening techniques.

FIG. 3A-C depicts photomicrographs of microarrays of the inventiongenerated within multichamber cell culture plates. (A) Shows a schematicof a microarray within a 96 well plate. (B) Shows a multi-well platewith subsequent magnification depicting the patterning of the cellulararray in each well. (C) Shows a micrograph of a co-culture of a firstcell type and a second cell type in the system of the invention.

FIG. 4 shows a method of the invention, whereby a microarray is preparedand seeded with cells that attach at discrete “spots” in the array. Thearray can then be treated or cultured under desired conditions and thenimaged or analyzed.

FIG. 5A-B shows the characterization of an ECM microarray by indirectimmunofluorescence. (A) Shows the composition and layout of each row. 32conditions in 8 replicates each were used. The spotting solutionconcentration of each ECM molecule, when present in a mixture, was 100μg/mL. (B) Shows the correlation of specified array compositions andimmunofluorescence of replicate arrays demonstrates presence andimmunoreactivity of all 5 ECM components with minimal carryover betweenconditions. C1, collagen I; C3, collagen III; C4, collagen IV; L,laminin; Fn, fibronectin.

FIG. 6A-D shows primary rat hepatocytes on ECM microarrays. (A) Shows aHoffman contrast montage image of the ECM microarray after 24 hours ofculture in 10% serum (magnified view in inset). Hepatocytes are wellspread, have bright intercellular borders and distinct nuclei, andspread to occupy the full ECM island in culture conditions. (B)Live/dead stains of hepatocytes using Calcein AM/ethidium homodimerdepicts ˜95% viability at 24 hours (magnified view in inset). Scalebars, 1 mm (inset scale bars, 500 μm). (C) High magnification phasecontrast and fluorescent images. (D) A single island (Calcein AM, DAPI).Scale bars, 50 μm.

FIG. 7A-D shows cultured hepatocytes demonstrate differentialintracellular albumin staining in response to matrix composition. (A)Indirect immunofluorescence of intracellular albumin, a marker ofdifferentiated hepatocyte function, on day 7. Note preservation ofmicroarray features after 7 days in 10% serum. (B) Quantitation ofaverage pixel intensity per microarray spot in panel A. (C) Hierarchicaldepiction of image albumin intensity for each of the matrix mixtures onday 7. Error bars represent s.e.m. (N=8). Reference line is the averageintensity for hepatocytes on day 1. Error box represents 1 s.d. (D)Results of 25 full factorial analysis on intracellular albumin intensity(4 microarray data sets). The relative magnitude of main effects as wellas 2-, 3-, 4-, and 5-factor interactions are shown. Q1, quadrant 1; Q2,quadrant 2; Q3, quadrant 3; Q4, quadrant 4; C1, collagen I; C3, collagenIII; C4, collagen IV; L, laminin; Fn, fibronectin.

FIG. 8A-E demonstrates that I114 embryonic stem cells differentiate onECM microarrays. (A) Bright field alkaline phosphatase staining of day 1ES cultures on ECM microarrays in 15% serum media (scale bar, 1 mm). (B)Phase contrast images of day 3 arrays cultured with LIF, and with RA(C). Cells cultured with LIF showed 3-dimensional features (see (B)inset x-z confocal section, ˜77 μm thickness). In contrast, RA-inducedcells grew as a relatively thin sheet (see (C) and inset x-z section,˜μm thickness). Scale bars, 250 μm (inset scale bars, 50 μm). (D) (topleft images) Bright field micrograph of selected X-gal stained ECMmicroarray conditions after 3 days of culture in RA. C1+C3+L+Fn inducedhigher levels of Ankrd17 (gtar) reporter activity (arrowheads) thancells cultured on C3+L (bottom left images). Scale bars, 250 μm.Magnified views of reporter activity: scale bars, 50 μm. Bar graph:hierarchical depiction of “blue” image area (pooled data from 4microarrays) for each of the matrix mixtures. Error bars represents.e.m. (N=32). The C1+C3+L+Fn culture condition induced ˜27-fold moreβ-galactosidase image area than the C3+L cultures. (E) Results of 25full factorial analysis on β-galactosidase positive “blue” image area (4microarray data sets). The relative magnitude of main effects as well as2-, 3-, 4-, and 5-factor interactions are shown. C1, collagen I; C3,collagen III; C4, collagen IV; L, laminin; Fn, fibronectin.

FIG. 9A-C show photomicrographs of hepatocyte cultures. (A) Primaryhepatocytes attach to collagen I spots, and are spatially confined onthe custom acrylamide, CodeLink (Amersham), and Hydrogel (Perkin Elmer)substrates. Superaldehyde (Telchem) and Epoxy Hydrogel (NoAbDiagnostics) allow cell attachment in non-spotted regions. (B) Primaryhepatocytes attached to collagen I spots made from solutions containingas little as 15.6 μg/mL of protein. Below this concentration non cellattachment was observed. Hepatocytes showed similar attachment toserially diluted collagen III, collagen IV, Laminin, and fibronectin.(C) Immobilized FITC-collagen I signal is linear over a broad range ofspotting solution protein concentrations. Error bars represent S.E.M.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a microwell”includes a plurality of such microwells and reference to “the cell”includes reference to one or more cells known to those skilled in theart, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The diversity of biological niches mandates a systematic approach toinvestigating optimal cell culture environments. Miniaturized arrays ofliving cells, like DNA microarrays, offer the potential of a more globalpicture of the role of soluble and insoluble cues on cell fate andfunction.

The cellular microenvironment plays a critical role in determining cellfate and function. Extracellular determinants of survival,proliferation, migration, and differentiation include soluble signals(cytokines, dissolved gasses), insoluble cues (extracellular matrix,cell-cell interactions, biomaterials), and physical stimuli (shearstress). Miniaturization of bioassays using multiwell plates and roboticliquid handling enables combinatorial screening of the effects ofsoluble species on cellular behavior; however, analogous approaches forscreening the effects of insoluble cues are in their infancy. Cellularinteractions with the extracellular matrix (ECM) are of particularinterest as ligation of an integrin can directly induce cellularsignaling, modulate the response to other agonists, and influence thebehavior of other integrins, a phenomenon called crosstalk. Thus, theextracellular matrix plays a role in developing an integrated picture ofthe microenvironment in the fate of many diverse cell types.

Cell-ECM interactions have been studied using several approaches.Typically, purified matrix proteins are adsorbed to cell culturesubstrates alone or in a combination requiring on the order of 10 μg ofprotein per 96-well plate; however, purified matrix for a combinatorialscreen can be prohibitively expensive and/or simply unavailable insufficient quantity. These ‘2-dimensional’ approaches are complementedby so-called ‘3-dimensional’ approaches such as embedding cells withinECM gels. More complex ECM has also been investigated using cell-derivedmatrix in vitro or decellularized tissue sections. In addition tonatural ECM components, biomaterial approaches have yielded severalhybrid matrices with tethered biomolecules and tunable degradation in a3-dimensional hydrogel context. Studies of the interaction of cell-ECMprovides a critical first step towards developing a comprehensiveunderstanding of insoluble cues in the cellular microenvironment.

Growth factor signals synergistically interact in permissive ECMmicroenvironments. Cross talk between ECM proteins and soluble factorswould be best investigated using a highly parallel microfluidic platformintegrating robotic spotting of substrates with a microfluidic networkgenerating combinatorial soluble factor mixtures. Such a platform can beused in other experiments to investigate other cellular pathwaysinvolving multiple soluble factor interactions and integrin cross talk.Microfluidics allows for on-chip serial dilution of soluble factorsthrough the exploitation of laminar flow inside the microchannels asshown in FIG. 2D-F. In addition, these dilutions can be performed onmultiple soluble factors and combined on-chip before delivery todiscreet microdomains of ECM proteins to study the combined effect ofsoluble and insoluble factors on cell fate and function. A schematic ofthis is shown in FIG. 2.

The invention provides a robust method to create cell arrays using aspotter device (e.g., a DNA spotter device) and off-the-shelf chemicals.Culturing parenchymal cells (e.g., primary hepatocytes), ES cells,and/or stromal cells on combinatorial mixtures of extracellular matrix(ECM) in the methods and systems of the invention yields novel insightsinto the role of the microenvironment using 1000 times less protein thanconventional methods. The methods and systems of the invention areamenable to depositing almost any insoluble or solublematerial/biological material, such as polysaccharides, proteoglycans,glycosaminoglycans, membrane bound proteins, DNA, siRNA, and tetheredgrowth factors or peptide signaling motifs. The methods and systems canalso be easily adapted to: exploit lineage-specific fluorescent reporterstrategies, co-cultivation of epithelia and stroma, and/or combinationsof soluble factors to screen the effects of growth factors or smallmolecules in conjunction with underlying matrix.

Referring to FIG. 1, there is shown a microarray 5 of the invention.Microarray 5 comprises a biologically compatible culture substrate 10having a first surface 12 and a second surface 14. The substrate 10 cancomprise a plurality of spots 50 a or microwells 50 b. Alternatively,the substrate 10 can be layered with a gel pad 20. Gel pad 20 may befabricated by polymerization of the gel using a template surface plate25 comprising protuberances 30 such that upon polymerization and removalof the surface plate 25 microwells 50 are formed in the gel pad 20. Inanother aspect, the gel pad 20 is polymerized and microwells 50 areformed using an etching technique, as described herein. Microwells 50can be any appropriate size but are typically about 100-200 μm indiameter. A spotter device 40 can be used to deliver the etchingmaterial. In addition, spotter device 40 can be used to deliver solubleor insoluble biological material 60 to microwells 50.

Various culture substrates 10 can be used in the methods and systems ofthe invention. Such substrates include, but are not limited to, glass,polystyrene, polypropylene, stainless steel, silicon and the like. Thechoice of the microarray surface should be taken in to account wherespatially separated cellular islands are to be maintained.

The cell culture surface (e.g., the first surface 12) can be chosen fromany number of rigid or elastic supports. For example, cell culturematerial can comprise glass or polymer microscope slides that have aplurality of relatively larger culture domains delineated by hydrophobicink, silicone well dividers, 3D surface topography, or a combination ofthese. Protein spots can thus be deposited, and cells cultured in eachculture well in a manner similar to that described herein.

The cell culture surface/substrate used in the methods and systems ofthe invention can be made of any material suitable for culturingmammalian cells. For example, the substrate can be a material that canbe easily sterilized such as plastic or other artificial polymermaterial, so long as the material is biocompatible. Substrate can be anymaterial that allows cells and/or tissue to adhere (or can be modifiedto allow cells and/or tissue to adhere or not adhere at selectlocations) and that allows cells and/or tissue to grow in one or morelayers. Any number of materials can be used to form thesubstrate/surface, including, but not limited to, polyamides;polyesters; polystyrene; polypropylene; polyacrylates; polyvinylcompounds (e.g. polyvinylchloride); polycarbonate (PVC);polytetrafluoroethylene (PTFE); nitrocellulose; cotton; polyglycolicacid (PGA); cellulose; dextran; gelatin, glass, fluoropolymers,fluorinated ethylene propylene, polyvinylidene, polydimethylsiloxane,polystyrene, and silicon substrates (such as fused silica, polysilicon,or single silicon crystals), and the like. Also metals (gold, silver,titanium films) can be used.

When certain materials such as nylon, polystyrene and similar materialsare used as the substrate it is advisable to pre-treat the substrateprior to inoculation with cells in order to enhance the attachment ofcells to the substrate. For example, prior to inoculation with stromalcells and/or parenchymal cells, nylon substrates should be treated with0.1M acetic acid, and incubated in polylysine, FBS, and/or collagen tocoat the nylon. Polystyrene could be similarly treated using sulfuricacid.

Additionally, rigid surfaces modified with a hydrogel can also be used.In one embodiment, user defined ECM protein mixtures are deposited on ahydrogel modified surface that is otherwise non-adhesive for cells. Thesurface may be a modified surface including, for example, a surface thathas been modified or coated with a gel pad 20. For example, in oneaspect of the invention, a substrate 10 is coated with polyacrylamidegel such that the surface allows ECM materials to be stored locally in ahydrated microwell environment, yet resists adsorption of serumproteins. The non-fouling nature of acrylamide prevents cell migrationover relatively long periods of time (˜28 days) in comparison to othercell patterning techniques (˜7 days) which use agarose, pluronics, serumalbumin, or polyethylene glycol. Dehydrated polyacrylamide substratesare thought to swell during spotting and retain proteins throughhydrophobic interactions. In order to maintain attachment of the gelmaterial to a substrate surface, the surface (e.g., glass) can bedensely hydroxylated prior to silanization, a highly porous glasssubstrate, or alternate silane coupling agents.

Alternatively, the hydrogel can be modified such that cell attachment isinhibited for a short period of time. During this period, a first celltype can be attached to the protein spotted domains. A second cell typecan then be introduced, which can attach to regions surrounding thefirst cell type. As a further embodiment, the invention provides for thecreation of polymer hydrogel “wells” by using a spotter device todeposit an etching solution (such as a mild periodic acid solution) to ahydrogel surface containing a degradable component. These etched wellscan then be further modified individually by subsequently depositingprotein solutions or pre-polymer solutions (with or without proteins) tothe well locations using the DNA-spotter. Proteins or pre-polymersolutions can thus be immobilized using photo-gelation or chemicalcrosslinking. Additionally, degradable polymer matrices can beincorporated into the hydrogel substrate that would allow for localsustained release of soluble factors. Again, each well can be tailoredindividually using the above mentioned techniques.

Polymeric hydrogels and gel pads can be used in the methods and systemsof the invention to facilitate cellular attachment and localization(e.g., by forming microwells). In one aspect, microwells are formed in agel layer/pad on a substrate to retain biological molecules including,but not limited to, proteins, peptides, oligonucleotides,polynucleotides, polysaccharides, lipids and other biological molecules.

Deformable hydrogel can be used in the methods and systems of theinvention. Deformable hydrogels include polyacrylamide hydrogels. Insome embodiment, the hydrogel will comprise components that weaklyrepulse cells, thereby providing low background binding. In oneembodiment, the substrate comprises a polymerized mixture includingacrylamide and hydrophilic acrylates.

Typically hydrogels are selected such that specific binding to desiredspots or wells by the cells is promoted and non-specific binding isreduced. Those of skill in the art will understand that cells vary intheir ability to adhere to a substrate.

Hydrogels provide for hydration of bound cells, lack of diffusion ofinsoluble materials, low background binding of cells and free flow ofcells across the surface of the microarray due to weak cell repulsion.

The cell culture surface can be created on a deformable membrane whichallows for the cells to be actively stretched during culture. Themembrane can be composed of an elastomeric material (such as PDMS),hydrogel, or other such material. Membrane deformation can be controlledusing any of a multitude of suitable methods. These include MEMS motorsincorporated into the culture chip, and connected to the culturesubstrate and electroactive polymers that respond to electric field byundergoing a shape change. Examples of such materials includeelectrostrictive materials such as thin acrylate films with deformableelectrodes placed on both sides of the material. An applied electricfield causes the acrylate film to compress or expand, resulting in aconcurrent change in surface area such that the total volume of the filmremains constant. Such materials are know as “electrostrictive” in thefield of electroactive polymers. Methods for generating force anddeformation in a pliable material using electric fields(“electrophoretic”), or alternating non-uniform electric fields(“dielectrophoretic”) are also possible by using a material that isresponsive to such modes of excitation. As an example, the gel materialmay incorporate charged particles, or neutral particles that canexperience an induced dipole force in the presence of a uniform ornon-uniform electric field. The electric field can be generated byplacing the gel material on an electrode array, which can apply a staticelectric field, or an alternating electric field of the appropriatefrequency to induce a net force on the dielectric medium of the gel.

By deformable is meant that a deformable material is capable of beingdamaged by contact with a rigid instrument. Examples of deformablematerials include hydrogels, polyacrylamide, nylon, nitrocellulose,polypropylene, polyester films, such as polyethylene terephthalate, andthe like. Non-deformable materials include materials that do not readilybend, and include glass, fused silica, nanowires, quartz, plastics, e.g.polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, andblends thereof, and the like; metals, e.g. gold, platinum, silver, andthe like. In some aspect, a deformable material may be layered upon anon-deformable material.

Another example of hydrogels useful in the methods and systems of theinvention include polyvinylalcohol (PVA), physically cross-linked bypartial crystallization of the chain. Such hydrogels are described, forexample, in U.S. Pat. No. 4,663,358. Another example are hydrogels basedon segmented polyurethanes or polyureas, an example of which isdescribed in U.S. Pat. No. 5,688,855. In principle, polypeptide orpolysaccharide hydrogels could be used, an example of which is agaroseor cross-linked hyaluronic acid. Other hydrogels include partiallyhydrolyzed or aminolyzed polyacrylonitrile (PAN), an example of which isdescribed in U.S. Pat. Nos. 4,943,618; 4,107,121, and 5,252,692.Typically, substrates and hydrogels used in the invention are sufficientbiocompatible and do not release any harmful substances.

The gel layer/pad can be conveniently produced as thin sheets on a solidsubstrate (e.g., a glass slide, cell culture plate, and the like),typically by depositing a solution of acrylamide monomer, a crosslinkersuch methylene bisacrylamide, and a catalyst such asN,N,N′,N′-tetramethylethylendiamine (TEMED) and an initiator such asammonium persulfate for chemical polymerization, or2,2-dimethoxy-2-phenyl-acetophone (DMPAP) for photo-polymerization. Inone aspect, the gel layer/pad is formed between two glass surfaces(e.g., glass plates or microscope slides) using a spacer to obtain thedesired thickness of the polymeric gel. Generally, the acrylamidemonomer and crosslinker are prepared in one solution of about 4-5%acrylamide (having an acrylamide/bisacrylamide ratio of 19/1) inwater/glycerol, with a nominal amount of initiator added. The solutionis polymerized and crosslinked either by ultraviolet (UV) radiation(e.g., 254 nm for at about 15 minutes, or other appropriate UVconditions), or by thermal initiation at elevated temperature (e.g.,typically at about 40° C.). Following polymerization and crosslinking,the top glass slide is removed from the surface to uncover the gel. Theporosity of the gel is controlled by changing the amount of crosslinkerand the percent solids in the monomer solution.

In the fabrication of polyacrylamide gels polymeric hydrogel arrays ofthe invention (i.e., patterned gels), the acrylamide solution typicallyis imaged through a mask during the UV polymerization/cross-linkingstep. The top glass slide is removed after polymerization, and theunpolymerized monomer is washed away (developed) with water, leaving afine feature pattern of polyacrylamide hydrogel, which is used toproduce the cross-linked polyacrylamide hydrogel. Further, lithographictechniques known in the semiconductor industry can be used to generatepatterned gel structures. Light can be applied to discrete locations onthe surface of a polyacrylamide hydrogel to activate specified regionsfor the attachment of an oligonucleotide, an antibody, an antigen, ahormone, hormone receptor, a ligand or a polysaccharide on the surface(e.g., a polyacrylamide hydrogel surface) of a solid support (see, forexample, International Application, Publication No. WO 91/07087,incorporated by reference).

For hydrogel-based arrays using polyacrylamide, biomolecules can beprepared by forming an amide, ester or disulfide bond between thebiomolecule and a derivatized polymer comprising the cognate chemicalgroup. Covalent attachment of the biomolecule to the polymer is usuallyperformed after polymerization and chemical cross-linking of the polymeris completed.

In some instances, it may be desirable to have etched polymer wellsincorporated into the cell culture surface. Such surfaces have beendescribed in the literature, however they require photolithography forthe fabrication. While such a method could be used with this invention,other techniques can be used. The invention also provides for thecreation of etched polymer wells using only a DNA-spotter, or similardevice. In one embodiment, an acrylamide coated slide is created whereinN,N′-(1,2-Dihydroxyethylene)bis-acrylamide (DHEBA) is incorporated as acrosslinking agent in the polymer network instead of bis-acrylamide. TheDNA spotter can then be used to deposit a mild solution of periodic acidwhich will dissolve the acrylamide network locally, thus creating a“microwell” (see FIG. 2A). The well can then be further modified bydepositing proteins or pre-polymer solutions which can be gelled orimmobilized in situ.

Controlled-release of soluble factors from a degradable polymersubstrate has been demonstrated in the fields of drug delivery andbiomimetic engineered surfaces. Typically, soluble factors areimmobilized within a polymer matrix or hydrogel. As the matrix degrades,soluble factors are released into the environment. The degradation ofthe polymer, and thus the release kinetics, can be tailored by modifyingthe composition of the polymer or hydrogel. In one variation, growthfactors are incorporated into poly(lactide-co-glycolide) (PLGA)microspheres. The GF-laden microspheres are then incorporated into asuitable matrix, such as PEG-hydrogel, PLGA, or acrylamide. FIG. 2D-Fshows several possible implementations of this strategy in conjunctionwith ECM microarrays and microfluidic networks.

Either or both the spotted surface and microfluidic channel surface canbe made to incorporate active or passive electronic components, such asresistive heating elements, photo-diodes, light-emitting diodes, analogamplifiers, and digital processing. These components would allow forself contained cell-culture, and monitoring of cellular processes usingin situ detection methods. In the simplest embodiment, this isaccomplished by choosing silicon as the substrate of choice for eitheror both the spotted substrate and microfluidic channel substrate, andusing standard silicon microelectronic fabrication techniques. FIG. 3 eillustrates various concepts that could be incorporated into such adevice.

As mentioned herein, in some instances the substrate may be modified topromote cellular adhesion and growth. For example, a glass substrate maybe treated with protein (i.e., a peptide of at least two amino acids)such as collagen or fibronectin to assist cells in adhering to thesubstrate. In some embodiments, the proteinaceous material is used todefine (i.e., produce) a microarray. The microarray produced by theprotein serves as a “template” for formation of the cellular microarray.Typically, a single protein will be adhered to the substrate, althoughtwo or more proteins may be used to spot a substrate using a spotterdevice. Proteins that are suitable for use in modifying a substrate tofacilitate cell adhesion include proteins to which specific cell typesadhere under cell culture conditions. For example, hepatocytes are knownto bind to collagen. Therefore, collagen is well suited to facilitatebinding of hepatocytes. Other suitable proteins include fibronectin,gelatin, collagen type IV, laminin, entactin, and other basementproteins, including glycosaminoglycans such as heparin sulfate.Combinations of such proteins also can be used.

With regard to placing insoluble and/or soluble factors at specificlocations, various micro-spotting techniques using computer-controlledplotters or even ink-jet printers have been developed to spot suchfactors at defined locations. One technique loads glass fibers havingmultiple capillaries drilled through them with differentoligonucleotides loaded into each capillary tube. Microarray substrate,such as a glass microscope slide, is then stamped out much like a rubberstamp on each glass slide. Spotting techniques involve the preciseplacement of materials at specific sites or regions using automatedtechniques.

Conventional physical spotting techniques such as quills, pins, ormicropipettors are able to deposit material on substrates in the rangeof 10 to 250 microns in diameter (e.g., about 100 spots/microwells perwe of a 96 well culture plate). In some instances the density can befrom 400 to 10000 spots per square centimeter, allowing for clearancebetween spots. Lithographic techniques, such as those provided byAffymetrix (e.g., U.S. Pat. No. 5,744,305, the disclosure of which isincorporated by reference herein) can produce spots down to about 10microns square, with no clearance between spots, resulting inapproximately 800,000 spots per square centimeter.

In some embodiments, materials (e.g., insoluble and/or solublematerials) are delivered (e.g. spotted) into at least one of theplurality of microwells in very small, e.g. nanoliter, increments usinga spotting device. The spotting device may employ one or morepiezoelectric pumps, acoustic dispersion, liquid printers, micropiezodispensers, or the like to deliver such reagents to each of themicrowells. In some embodiments, the spotting device comprises anapparatus and method like or similar to that described in U.S. Pat. Nos.6,296,702, 6,440,217, 6,579,367, and 6,849,127.

Accordingly, an automated spotting device can be utilized, e.g. PerkinElmer BioChip Arrayer™. A number of contact and non-contact microarrayprinters are available and may be used to dispense/print the solubleand/or insoluble materials on a substrate. For example, non-contactprinters are available from Perkin Elmer (BioChip Arrayer™), Labcyte andIMTEK (TopSpot™), and Bioforce (Nanoarrayer™). These devices utilizevarious approaches to non-contact spotting, including piezo electricdispension; touchless acoustic transfer; en bloc printing from multiplemicrochannels; and the like. Other approaches include ink jet-basedprinting and microfluidic platforms. Contact printers are commerciallyavailable from TeleChem International (Arraylt™). Non-contact printersare of particular interest because they are more compatible withdeformable hydrogel surfaces and allow for simpler control over spotsize via multiple dispensing onto the same location.

Non-contact printing will typically be used for the production ofcellular microarrays. By utilizing a printer that does not physicallycontact the surface of substrate, no aberrations or deformities areintroduced onto the substrate surface, thereby preventing uneven oraberrant cellular capture at the site of the spotted material. Suchprinting methods find particular use with hydrogel substrates.

Printing methods of interest, including those utilizing acoustic orother touchless transfer, also provide benefits of avoiding clogging ofthe printer aperature, e.g. where probe solutions have high viscosity,concentration and/or tackiness. Touchless transfer printing alsorelieves the deadspace inherent to many systems. The use of print headswith multiple ports and the capacity for flexible adjustment of spotsize can be used for high-throughput microarray preparation.

The total number of spots on the substrate will vary depending on thenumber of different conditions (e.g., material combinations) to beexplored, as well as the number of control spots, calibrating spots andthe like, as may be desired. Generally, the pattern present on thesurface of the support will comprise at least 2 distinct spots, usuallyabout 10 distinct spots, and more usually about 100 distinct spots,where the number of spots can be as high as 50,000 or higher. Thespot/microwell will usually have an overall circular dimension and thediameter will range from about 10 to 5000 μm (e.g., about 20 to 1000μm).

By dispensing or printing onto the surfaces or into a microwell ofmulti-well culture plates, one can combine the advantages of the arrayapproach with those of the multi well approach. Typically, theseparation between tips in standard spotting device is compatible withboth a 384 well and 96 well plate, one can simultaneously print eachload in several wells. Printing into wells can be done using bothcontact and non-contact technology (as described above).

The methods and systems of the invention are useful to modulate thedensity of biological materials “spotted” on a cell culture substrate.For example, the maximum density of ECM molecules is dependent onseveral factors including: the concentration of stock solution, thesolubility of ECM proteins, the porosity of the substrate or gel (e.g.,polyacrylamide gel), and the mode of deposition (e.g., pin orpiezoelectric). Controlling the amount/density of biological materialsin a culture environment can modulate cell growth and differentiation.For example, a minimum surface density of integrin ligands is requiredfor cell attachment and spreading, and is estimated to be as low as 1ng/cm² for hepatocyte cells on laminin, fibronectin, collagen I, andcollagen IV. Accordingly, the spotting device can be calibrated and usedto provide specific amounts of insoluble and/or soluble biologicalmaterials to select “spots” or microwells.

The invention provides methods and systems useful for identifyingoptimal conditions for controlling cellular development and maturation.For example, the methods and systems of the invention are useful foridentifying optimal conditions that control the fate of cells (e.g.,differentiating stem cells into more mature cells, maintenance ofself-renewal, and the like) by controlling and optimizing theextracellular and soluble microenvironment upon which the cells arecultured in parallel array fashion for rapid high throughput techniques.

A miniaturized cell culture microarray platform of the invention isuseful for testing a multitude of soluble factors (e.g., growth factors,hormones, steroids and the like) and insoluble factors (e.g.,extracellular matrix, cell adhesion proteins, glycoproteins and thelike) individually and in combination using minimal reagents and arelatively small numbers of cells.

The invention utilizes robotic spotting technology to develop a robust,accessible method for forming cellular microarrays on, for example, anadherence material such as combinatorial extracellular matrixdomains—that required no photolithographic ‘cell micropatterning’ toolsor custom-built equipment and only small amounts of protein (˜10 pg) perexperimental condition. As used herein, the term “microarray” refers toa plurality of addressed or addressable locations (e.g., microwells).The location of each of the microwells or groups of microwells in thearray is typically known, so as to allow for identification and, as morefully described below, assay of particular changes in expression,morphology, and the like.

With the advent of DNA robotic spotting technology, it is now possibleto routinely deliver nanoliter volumes of many different materials, frominterfering RNA, to peptides, to biomaterials at precise locations on amicroarray substrate. To date, techniques that use spotted microarraysfor cell culture have not been appropriate for manipulation of ECMmaterial due to, for example, incompatible process conditions for ECMprotein spotting, extensive customization of spotting equipment, or lackof pattern fidelity (i.e. cell localization) over time.

In one aspect, the invention provides methods and systems that overcomethese limitations by, for example, modifying the printing buffer used ina spotting device to allow for ECM deposition, and identifyingmicroarray substrates that permit ECM immobilization. For example, inone aspect, the substrate is a hydrogel surface that maintains spatiallyconfined cellular islands through the use of microwell generation in ahydrogel material.

The methods and systems of the invention are useful for spottingsubstantially purified or mixtures of biological proteins, nucleic acidsand the like (e.g., collagen I, collagen III, collagen IV, laminin, andfibronectin) in various combinations on a standard cell culturesubstrate (e.g., a microscope slide) using off-the-shelf chemicals and aconventional DNA robotic spotter (FIGS. 1 and 3).

Cell culture is a highly empirical field, thus this platform allows fora multitude of insoluble factors (e.g. extracellular matrix,biomaterials), tethered soluble factors (e.g., growth factors), ormixtures of insoluble and soluble cues to be tested in parallel on asmall scale.

The term “adherence material” is a material deposited on a substrate orchip or within a microwell in an array for which a cell or microorganismhas some affinity, such as a binding agent. The material can bedeposited in a domain, microwell or “spot”. The material and a cell ormicroorganism interact through any means including, for example,electrostatic or hydrophobic interactions, covalent binding or ionicattachment. The material may include, but is not limited to, antibodies,proteins, peptides, nucleic acids, peptide aptamers, nucleic acidaptamers, or cellular receptors.

A “plurality of domains” or “spots” or “microwells” includes more thanone discrete domain or spot of an adherence material that is depositedonto a solid support, or a material that is deposited onto the surfaceof a solid support. In one embodiment the solid support and the surfacewill form a chip. Different materials that specifically interact withdifferent cells or microorganisms may be deposited in separate anddiscrete domains on or in the substrate surface.

In one embodiment, the invention provides a surface comprising multipledistinct cell culture domains (microwells) of arbitrary protein orpolymer composition and size by using robotic spotting technology (e.g.,a DNA spotting device or a similar device), to transfer nanoliterquantities of material onto a culture substrate (e.g., glass, silicon,polymer or other biocompatible material used in cell culture) at desiredlocations.

In one embodiment, one or more desired biological materials aredeposited as discrete “spots” on a culture substrate surface. In oneaspect, each spot comprises a different biological material composition.In another aspect, each spot comprises the same biological materialcomposition. Cells cultured on the spots may be the same or different.For example, a defined ECM material is deposited as discrete spots ontoa culture substrate surface. Cells are then contacted with the substrateand cultured under desired culture conditions. Where the spots comprisedifferent biological materials, the cells experience different stimuliwhile being cultured simultaneously but maintained in distinct spatialdomains creating a cellular array.

The spotted surface can also be bonded with a molded polymer or etchedsurface such that a microfluidic network is created with a plurality ofchannels and chambers. The fluidic network can be engineered to mixsoluble factors and deliver them to addressable locations on themicroarrayed surface. This would allow for cell culture media additivesto be modulated in an efficient manner as only small volumes would berequired.

A microfluidic network can be fabricated through micromolding of anelastomer against a negative relief of the microchannels, or byselectively etching a surface of a material such as glass or silicon.This network is bonded to the protein microspotted substrate. Themicrochannels can be designed to deliver specific concentrations andcombinations of soluble factors to various locations on the array.

Either or both the spotted surface and microfluidic channel surface canbe made to incorporate active or passive electronic components, such asresistive heating elements, photo-diodes, light-emitting diodes, analogamplifiers, and digital processing. These components would allow forself contained cell-culture, and monitoring of cellular processes usingin situ detection methods. In the simplest embodiment, this isaccomplished by choosing silicon as the substrate of choice for eitheror both the spotted substrate and microfluidic channel substrate, andusing standard silicon microelectronic fabrication techniques.

Microfluidic channels may be used in the methods of the invention todeliver media and/or reagents to cells in microwells 50. Themicrofluidic channels may be part of the substrate 10 or may be furtheretched or coated onto gel pad 20 (see, e.g., FIGS. 2D-F). Typically, thesystem comprises a culture microwell having an inlet and an outletdesigned to allow fluid to flow across/through the microwell. Cellsand/or biological material (e.g., ECM) can be disposed between and influid communication with the inlet and outlet. The at least one inletand one outlet can comprise valves to control fluid flow.

Thus, devices of the invention can include at least one flow channelthat allows fluid flow from an inlet to an outlet. As will beappreciated by those in the art, the flow channels may be configured ina wide variety of ways, depending on the use of the channel. Forexample, a single flow channel may be separated into a variety ofsmaller or similarly sized channels, such that the original sample isdivided into discrete subsamples for parallel processing or analysis(see, e.g., FIG. 2D-F). Alternatively, several flow channels may feedtogether into a single channel. As will be appreciated by those in theart, there are a large number of possible configurations; what isimportant is that the flow channels allow the movement of sample andreagents from one part of the array to another.

In another aspect of the invention, the system comprises at least onepump. These pumps can be any type of pump device including electrodebased pumps. Electromechanical pumps can be used in the systems of theinvention, e.g. based upon capacitive, thermal, and piezoelectricactuation. Suitable “on chip” pumps include, but are not limited to,electroosmotic (EO) pumps and electrohydrodynamic (EHD) pumps; theseelectrode based pumps have sometimes been referred to in the art as“electrokinetic (EK) pumps”. In another aspect, the pumps are externalto the microfluidic channel. In this aspect, the pump may be aperistaltic pump, syringe pump or other pump commonly used in the art.

A microfluidic flow regulator can be used in the system and methods ofthe invention, such as one or more of the micropumps described herein,for controlling the flow rate. Such pumps may include, for example, amicroelectromechanical (MEMS) microfluidic pump. The micropump can beoperated at a predetermined frequency, which can be either substantiallyconstant or modulated depending upon the requirements of the system(e.g., the cellular metabolism, metabolite delivery and the like).

In another aspect of the invention, microarray devices of the inventionmay include at least one fluid valve that can control the flow of fluidinto or out of a microarray device or divert the flow into one or morechannels. A variety of valves are known in the art. For example, in oneembodiment, the valve may comprise a capillary barrier.

In yet another embodiment, sealing ports may be used to allow theintroduction of fluids, including samples, into the array of theinvention, with subsequent closure of the port to avoid the loss of thesample.

The devices of the invention can include at least one storage modulesfor assay reagents (e.g., buffer, sample, and culture media). These maybe fluidly connected to other modules of the system using flow channelsand may comprise wells or chambers, or extended flow channels.

The microfluidics can be utilized to deliver test analytes to cellularmicroarrays of the invention. Analytes include organic and inorganicmolecules, including biological molecules. For example, the analyte maybe an environmental pollutant (including pesticides, insecticides,toxins, and the like); a chemical (including solvents, polymers, organicmaterials, and the like); therapeutic molecules (including therapeuticand abused drugs, antibiotics, and the like); biological molecules(including, e.g., hormones, cytokines, proteins, lipids, carbohydrates,cellular membrane antigens and receptors (neural, hormonal, nutrient,and cell surface receptors) or their ligands); whole cells (includingprokaryotic and eukaryotic cells; viruses (including, e.g.,retroviruses, herpesviruses, adenoviruses, lentiviruses); spores; andthe like.

Regarding the cell culture aspect, one cell type of a plurality of celltypes can be used in the microarrays of the disclosure. For example,cultures of two cell types can be performed thus allowing for co-cultureof two cell types. For example, the culture surface surrounding the afirst spot can be modified such that cells cannot attach to or migrateto the regions surrounding the spot. In this manner, mono-culture orco-culture involving multiple cell types and multiple ECM coatings canbe tested in a single 96-well plate.

The type of biological materials (e.g., ECM materials) deposited in themicroarray will be determined, in part, by the cell type or types to becultured. For example, ECM molecules found in the hepaticmicroenvironment are useful in culturing hepatocytes, the use of primarycells, and a fetal liver-specific reporter ES cell line. The liver hasheterogeneous staining for collagen I, collagen III, collagen IV,laminin, and fibronectin. Hepatocytes display integrins β1, β2, α1, α2,α5, and the nonintegrin fibronectin receptor Agp110 in vivo. Culturedrat hepatocytes display integrins α1, α3, α5, β1, and α6β1, and theirexpression is modulated by the culture conditions. It will be recognizedthat the initial material used in the array will be modulated bycellular interactions. For example, interaction with ECM is known tomodulate matrix metalloproteinase expression, integrin activity, andmatrix expression.

ECM and growth factor interactions, which can be positive or negative,have been reported in multiple contexts. In addition to the describedcrosstalk between integrin and growth factor signaling, crosstalk existsamong matrix molecules. For example, it has been reported that collageninduced release of IL-1 through binding integrin α7β1 in human bloodmononuclear cells is potentiated by fibronectin binding to α5β1.Similarly, endothelial cell attachment to fibronectin via α5β1 integrinreportedly potentiates αvβ3-mediated migration on vitronectin. In thiscontext, the invention and the results presented below with hepatocytessuggest that integrin crosstalk may explain the apparent antagonisticinteractions among collagen III and laminin, and collagen IV andfibronectin; a hypothesis that is testable by the methods and systems ofthe invention.

The invention is advantageously used for performing assays onmicrochips/biochips. Microchips (also sometimes referred to as biochips)encompass substrates containing arrays or microarrays, typically orderedarrays and most commonly ordered, addressable arrays, of a biologicalmaterial or a mixture of biological materials.

One useful feature of such chips is the manner in which the arrayedbiomolecules are attached to the surface of the chip's substrate.Conventionally such procedures involve reaction steps, includingchemical modification of the solid support itself. In some aspects,modification of the support surface or modification of the gel polymeris useful to provide a chemical functionality capable of formingsufficient bonding.

The effect of soluble and insoluble factors/test analytes have on cellsin a microarray can be probed for a specific marker, examined formorphology, and the like. For example, the array can be probed for thestate of differentiation using various techniques including in situhybridization, antigenic recognition (intracellular or cell membrane),in situ PCR, or an artificial DNA-reporter construct such as GFP orbeta-galactosidase. The cell array can be assessed using fluorescentmicroscopy, high resolution light microscopy, a confocal laser scanner(such as those commonly used for DNA microarray applications),fluorescence and absorbance plate readers, scanners, or other suchequipment. In many aspect, the measurements will be made by automatedmicroscopes, plate readers and the like. In this manner, “optimal”culture conditions, as defined by the user, can be identified for closerexamination and testing using more conventional techniques.

Fixation of cells generally leads to disruption of cellulararchitecture, loss of antigen recognition, and loss of soluble matterfrom “leaky” cells during the numerous washing steps. As an alternative,cellular arrays can be freeze transferred to a nitrocellulose membrane,a process known in the field as “cytocoherent transfer”. This processpreserves the spatial (X-Y) distribution of proteins, but significantlycompresses the 3D data into a more 2D configuration. The proteins aremuch better retained by the nitrocellulose membrane, which also providesexcellent access to the antigenic sites that can be probed usingimmunochemical detection methods. Cytotransfer is accomplished bycryogenically freezing the cell culture array, followed by placing it incontact with a thin nitrocellulose (or other material) membrane. Thisact initiates a “thaw transfer” of the biological material to themembrane. This process is advantageous for several reasons: it does notrequire fixatives, small cytosolic molecules are directly transferred tothe membrane (whereas they might be washed away during fixation andwashing), the membrane provides excellent antigenic access, and the 3Dspatial protein information is somewhat compressed into a more 2D, butaccurate in the planar direction, representation. Compressing theinformation into a more 2D format is advantageous from a quantificationstandpoint. Typically confocal laser scanners are used to quantifymicroarray format material. The 3D nature of cell cultures presents adifficulty for such equipment, unless multiple z-planes are imaged andanalyzed. Often, the 3D information is not of practical value, and a 2Drepresentation would be sufficient. Compressing the 3D cellular proteinpresentation into a more 2D representation prior to detection thusalleviates the need to image multiple z-planes using a such a confocaldevice. Alternatively, the same technique can be used in conjunctionwith luminescent assays simply because of the advantage of improvedcellular protein retention in the membrane, which results in anincreased signal. Such techniques are further described in McGrath etal., BioTechniques 11:352-361, 1991.

Cells cultured on microarrays of the disclosure may be used to studycell and tissue morphology. For example, enzymatic and/or metabolicactivity may be monitored in the culture by fluorescence orspectroscopic measurements on a conventional microscope. In one aspect,a fluorescent metabolite in the fluid/media is used such that cells willfluoresce under appropriate conditions (e.g., upon production of certainenzymes that act upon the metabolite, and the like). Alternatively,recombinant cells can be used in the cultures system, whereby such cellshave been genetically modified to include a promoter or polypeptide thatproduces a therapeutic or diagnostic product under appropriateconditions (e.g., upon zonation or under a particular oxygenconcentration). For example, a hepatocyte may be engineered to comprisea GFP (green fluorescent protein) reporter on a P450 gene (CYPIA1).Thus, if a drug activates the promoter, the recombinant cell fluoresces.This is useful for predicting drug-drug interactions that occur due toupregulation in P450s.

Embryonic stem cells are a potential source of differentiated cells thatcould be used in cell therapy, drug discovery, and basic research.Current methods for differentiating embryonic stem cells in vitro aregenerally inefficient (˜1%) for generating specific lineages, and relyon the use of heterogeneous cell aggregates called embryoid bodies.Exceptions to this generalization are a few rare reports of efficientmonolayer culture methods, underscoring the importance of a tightlyregulated environment for efficient lineage-specific differentiation.While most studies focus on growth factors, the importance of ECM indevelopmental processes has increasingly been recognized. In vitro,undifferentiated mouse ES cells express integrins α6, β1, β4, β5,laminin receptor 1, and dystroglycan; thus poised to receive signalsfrom ECM. Given that stem cell differentiation has historically been alargely empirical field, a parallelized culture platform is of benefit.Monitoring can be performed using specific markers or ubiquitouscellular constituents such as actin and keratin.

In one aspect, the microarray utilizes ECM protein microarrays to studyand obtain ES differentiation in the context of the liver. In vitrodifferentiation of I114 mouse embryonic reporter stem cells as embryoidbodies induces reporter expression that coincides with endodermal geneupregulation, and co-localization with alpha-fetoprotein and albuminprotein. As such, it serves as a tool to study early hepaticspecification in vitro. Culturing ES cells as 3-dimensional aggregatesgreatly improves the frequency of differentiation to somatic lineages;however, embryoid bodies are seldom uniform in size. The inventionprovided methods that produce uniform, spatially confined, 3-D growth ofES cells cultured on ECM microarrays with LIF. The microarray provides aconfined domain that result in appropriate differentiation and growth.In contrast, culturing with RA resulted in a noticeable flattening ofthe cellular island morphology likely due to induction ofdifferentiation, a reduction in proliferation, and therefore lessexpansion in the z-direction. From image analysis reporter activity inday 9 EB's was estimated at <1% and even less in monolayer cultures. Incontrast, the day 3 RA-induced micro-cultures exhibited up to 16.8%±5.5%(N=8) of the island area showing reporter activity when cultured onoptimal ECM microenvironments. Moreover, an approximate 140-foldincrease was detected in β-galactosidase signal from the least efficientcondition (laminin only) to the most efficient (laminin+collagenI+fibronectin). Thus, the invention provides methods and systems toinvestigate and identify how complex ECM enhance reporter activityproviding valuable insight on how to drive in vitro differentiation moreefficiently.

The invention described herein provides a substrate that can be read oranalyzed by a variety of methods including, but not limited, tofluorescence, surface plasmon resonance, mass spectrometry, quartzcrystal resonance, electron microscopy and scanning probe microscopy. Inone aspect scanning probe microscopy (SPM) such as atomic forcemicroscopy (AFM). Use of an AFM or another type of SPM creates amethodology for a simple rapid, sensitive and high throughput method fordetection of microorganisms, pathogens, biological matter, viruses, ormicroparticles (Moloney et al., 2002, Ultramicroscopy 91 pp. 275-279).This method can be used to detect changes in a spot sample.Additionally, fluorescence or other methods commonly practiced fordetection of biological events can be employed in the methods andsystems of the invention.

The invention provides technology that can be useful for a variety ofpurposes, such as determining the appropriate culture conditions fordifferentiating stem cells into more mature cells, studying cell-matrixand growth factor interactions in a systematic manner, and potentiallyscreening new drug molecule candidates for their effects on cells invitro by immobilizing small volumes in degradable matrices for sustainedrelease. Additionally, the platform can be extended for use withnon-stem cells, such as primary cells (e.g. hepatocytes, fibroblasts),genetically modified cells, and transformed or cancerous cell types.

A number of uses of the methods and systems will be readily apparent toone of skill in the art. For example, stem cell therapeutic companiescould use such technology to optimize differentiation protocols forspecific lineages. Lifescience or pharmaceutical companies could usesuch technology for optimizing in vitro production of recombinantproteins. Pharmaceutical companies could use a miniaturized cell cultureplatform to test toxicity of potential drug compounds in a parallelmanner using minimal reagents. Researchers could use such a platform totest the effects of insoluble or tethered soluble and insoluble cues oncellular differentiation.

The culture system and microarrays of the disclosure can be used in awide variety of applications. These include, but are not limited to,screening compounds, growth/regulatory factors, pharmaceuticalcompounds, and the like, in vitro; elucidating the mechanisms of certaindiseases; studying the mechanisms by which drugs and/or growth factorsoperate; diagnosing and monitoring cancer in a patient; the productionof biological products, to name a few.

The methods and systems of the disclosure may be used to in vitro toscreen a wide variety of compounds, such as cytotoxic compounds,growth/regulatory factors, pharmaceutical agents, and the like, toidentify agents that modify cell (e.g., hepatocyte) function and/orcause cytotoxicity and death or modify proliferative activity or cellfunction. For example, the culture system may be used to testadsorption, distribution, metabolism, excretion, and toxicology (ADMET)of various agents. The activity of a compound can be measured by itsability to damage or kill cells in culture or by its ability to modifythe function of the cells (e.g., in hepatocytes the expression of P450,and the like). This may readily be assessed by vital stainingtechniques, ELISA assays, immunohistochemistry, and the like. The effectof growth/regulatory factors on the cells (e.g., hepatocytes,endothelial cells, epithelial cells, pancreatic cells, astrocytes,muscle cells, cancer cells) may be assessed by analyzing the cellularcontent of the culture, e.g., by total cell counts, and differentialcell counts or by metabolic markers such as MTT and XTT. This may alsobe accomplished using standard cytological and/or histologicaltechniques including the use of immunocytochemical techniques employingantibodies that define type-specific cellular antigens. The effect ofvarious drugs on normal cells cultured in the culture system may beassessed. For example, drugs that affect cholesterol metabolism, e.g.,by lowering cholesterol production, could be tested on a liver culturesystem.

The cytotoxicity to cells in culture (e.g., human hepatocytes) ofpharmaceuticals, anti-neoplastic agents, carcinogens, food additives,and other substances may be tested by utilizing the microfluidicmicroarrays of the invention.

The invention provides a screening method comprising, generating amicroarray on a substrate using a spotting device (e.g., a DNA spottingdevice) or similar device. A material spotted by the device may includesoluble and/or insoluble factors that have known activity or effects oncells or may comprise factors having unknown activity of effects oncells. Cells are then contacted with the micro-spot array and a stable,growing culture is established. The cells may then be examined oralternatively, the cells are exposed to varying concentrations of a testagent. After incubation, the culture is examined by phase microscopy todetermine the effect of the material and/or test agent on a cell'smorphology, growth, activity, and the like. Cytotoxicity testing can beperformed using a variety of supravital dyes to assess cell viability inthe liver culture system, using techniques well-known to those skilledin the art.

Similarly, the beneficial effects of drugs or biologics may be assessedusing the microarray system. For example, growth factors, hormones, ordrugs which are suspected of having the ability to enhance cell ortissue function, formation or activity can be tested. In this case,stable cultures are exposed to a test agent. After incubation, thecultures are examined for viability, growth, morphology, cell typing,and the like, as an indication of the efficacy of the test substance.Varying concentrations of the drug may be tested to derive adose-response curve.

The culture systems of the disclosure may be used as model systems forthe study of physiologic or pathologic conditions and to optimize theproduction of a specific protein. For example, in a specific embodiment,a parenchymal cell culture (e.g., a liver culture) system can beoptimized to act in a specific functional manner as described herein byspotting defined soluble and/or insoluble factors.

The microarray culture system may also be used to aid in the diagnosisand treatment of malignancies and diseases. For example, a biopsy of atissue (such as, for example, a liver biopsy) may be taken from asubject suspected of having a malignancy or other disease or disorder.The biopsy cells can then be cultured under appropriate conditions(e.g., defined factors spotted on an array) where the activity of thecultured cells can be assessed using techniques known in the art. Inaddition, such biopsy cultures can be used to screen agent that modifythe activity in order to identify a therapeutic regimen to treat thesubject. For example, the subject's tissue culture could be used invitro to screen cytotoxic and/or pharmaceutical compounds in order toidentify those that are most efficacious; i.e. those that kill themalignant or diseased cells, yet spare the normal cells. These agentscould then be used to therapeutically treat the subject.

Similarly, the beneficial effects of drugs may be assessed using amicroarray in vitro; for example, growth factors, hormones, drugs whichenhance hepatocyte formation or activity can be tested. In this case,microarray cultures may be exposed to a test agent. After incubation,the microarray cultures may be examined for viability, growth,morphology, cell typing, and the like as an indication of the efficacyof the test substance. Varying concentrations of the drug may be testedto derive a dose-response curve.

In one aspect, the study of hepatocyte function, differentiation, growthand metabolism can be examined. Isolated human hepatocytes are highlyunstable in culture and are therefore of limited utility for studies ondrug hepatotoxicity, drug-drug interaction, drug-related induction ofdetoxification enzymes, and other liver-based phenomena. The alternativeapproach is to employ animal experimentation to study the liver'sresponse; however, there are many well-documented differences betweenanimal and human metabolism that lead to inconclusive or inaccurateinterpretation of animal data for human applications. The disclosureprovides a micro-scale model of human liver cell and/or tissue that canbe utilized for pharmaceutical drug development, basic science research,and in the development of tissue for transplantation.

In one aspect, micropatterned cultures comprising parenchymal cells andstromal cells are used in the methods and systems of the invention. Forexample, islands of material (e.g., protein material—soluble orinsoluble) can be generated on a substrate to form an array. Each islandmay comprise the same material or an array of patterned differentmaterial. In some cases, adjacent islands comprise different materialsthat are directed to a particular cell type to be studied. In thismanner and array of cell-types may be simultaneous cultured. Forexample, the substrate is modified and prepared such that stromal cellsare interspersed with the parenchymal cells. Using a DNA spotter device,the substrate is modified to provide for spatially arranging parenchymalcells (e.g., human hepatocytes and supportive stromal cells (e.g.,fibroblasts)) in a miniaturizable format. Specifically, parenchymalcells (e.g., hepatocytes) can be prepared in islands surrounded bystromal cells (e.g., fibroblast such as murine 3T3 fibroblasts).Furthermore, parenchymal cell function may be modified by altering thepattern configuration.

Using micropatterning of co-cultures and reagents can lead to a cell ortissue model that can be optimized for specific physiologic functionsincluding, for example, synthetic, metabolic, or detoxification function(depending on the function of interest) in hepatic cell cultures.

In one aspect, the system utilizes co-cultures of cells in which atleast two types of cells are configured in a micropattern on asubstrate. By using micropatterning techniques to modulate the extent ofheterotypic cell-cell contacts. In addition, co-cultures (bothmicropatterned co-cultures and non-micropatterned co-cultures) haveimproved stability and thereby allow chronic testing (e.g., chronictoxicity testing as required by the Food and Drug Administration for newcompounds). Because micropatterned co-cultures are more stable thanrandom cultures the use of co-cultures and more particularlymicropatterned co-cultures provide a beneficial aspect to the culturessystems of the disclosure. Furthermore, because drug-drug interactionsoften occur over long periods of time the benefit of stable co-culturesallows for analysis of such interactions and toxicology measurements.

Typically, in practicing the methods of the disclosure, the cells aremammalian cells, although the cells may be from two different species(e.g., pigs, humans, rats, mice, and the like). The cells can be primarycells, stem cells, or they may be derived from an established cell-line.Although any cell type that adheres to a substrate can be used in themethods and systems of the disclosure (e.g., parenchymal and/or stromalcells), exemplary cell include, hepatocytes, epithelial cells,endothelial cells, pancreatic cells, muscle cells, neuronal cells, etc.

Cells useful in the methods and to populate a micro-spotted substrate ormicrowell of the disclosure are available from a number of sourcesincluding commercial sources. For example, hepatocytes may be isolatedby conventional methods (Berry and Friend, 1969, J. Cell Biol.43:506-520) which can be adapted for human liver biopsy or autopsymaterial. Typically, a canula is introduced into the portal vein or aportal branch and the liver is perfused with calcium-free ormagnesium-free buffer until the tissue appears pale. The organ is thenperfused with a proteolytic enzyme such as a collagenase solution at anadequate flow rate. This should digest the connective tissue framework.The liver is then washed in buffer and the cells are dispersed. The cellsuspension may be filtered through a 70 μm nylon mesh to remove debris.Hepatocytes may be selected from the cell suspension by two or threedifferential centrifugations.

For perfusion of individual lobes of excised human liver, HEPES buffermay be used. Perfusion of collagenase in HEPES buffer may beaccomplished at the rate of about 30 ml/minute. A single cell suspensionis obtained by further incubation with collagenase for 15-20 minutes at37° C. (Guguen-Guillouzo and Guillouzo, eds, 1986, “Isolated and CultureHepatocytes” Paris, INSERM, and London, John Libbey Eurotext, pp. 1-12;1982, Cell Biol. Int. Rep. 6:625-628).

Hepatocytes may also be obtained by differentiating pluripotent stemcell or liver precursor cells (i.e., hepatocyte precursor cells). Theisolated hepatocytes may then be used in the culture systems describedherein.

Stromal cells include, for example, fibroblasts obtained fromappropriate sources as described further herein. Alternatively, thestromal cells may be obtained from commercial sources or derived frompluripotent stem cells using methods known in the art.

Fibroblasts may be readily isolated by disaggregating an appropriateorgan or tissue which is to serve as the source of the fibroblasts. Thismay be readily accomplished using techniques known to those skilled inthe art. For example, the tissue or organ can be disaggregatedmechanically and/or treated with digestive enzymes and/or chelatingagents that weaken the connections between neighboring cells making itpossible to disperse the tissue into a suspension of individual cellswithout appreciable cell breakage. Enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withany of a number of digestive enzymes either alone or in combination.These include but are not limited to trypsin, chymotrypsin, collagenase,elastase, and/or hyaluronidase, DNase, pronase, dispase and the like.Mechanical disruption can also be accomplished by a number of methodsincluding, but not limited to, the use of grinders, blenders, sieves,homogenizers, pressure cells, or insonators. For a review of tissuedisaggregation techniques, see Freshney, Culture of Animal Cells. AManual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch.9, pp. 107-126.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thefibroblasts and/or other stromal cells and/or elements can be obtained.This also may be accomplished using standard techniques for cellseparation including, but not limited to, cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counter-streaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis, fluorescence-activatedcell sorting, and the like. For a review of clonal selection and cellseparation techniques, see Freshney, Culture of Animal Cells. A Manualof Basic Techniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11and 12, pp. 137-168.

The isolation of fibroblasts can, for example, be carried out asfollows: fresh tissue samples are thoroughly washed and minced in Hanksbalanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated fibroblasts can then be used in the culture systemsof the disclosure.

For example, and not by way of limitation, endothelial cells may beisolated from small blood vessels of the brain according to the methodof Larson et al. (1987, Microvasc. Res. 34:184) and their numbersexpanded by culturing in vitro using the bioreactor system of thedisclosure. Silver staining may be used to ascertain the presence oftight junctional complexes specific to small vessel endothelium andassociated with the “barrier” function of the endothelium.

Suspensions of pancreatic acinar cells may be prepared by an adaptationof techniques described by others (Ruoff and Hay, 1979, Cell Tissue Res.204:243-252; and Hay, 1979, in, “Methodological Surveys in BiochemistryVol. 8, Cell Populations.” London, Ellis Hornwood, Ltd., pp. 143-160).Briefly, the tissue is minced and washed in calcium-free, magnesium-freebuffer. The minced tissue fragments are incubated in a solution oftrypsin and collagenase. Dissociated cells may be filtered using a 20 μmnylon mesh, resuspended in a suitable buffer such as Hanks balanced saltsolution (HBSS), and pelleted by centrifugation. The resulting pellet ofcells can be resuspended in minimal amounts of appropriate media andinoculated onto a substrate for culturing in the bioreactor system ofthe disclosure. The pancreatic cells may be cultured with stromal cellssuch as fibroblasts. Acinar cells can be identified on the basis ofzymogen droplet inclusions.

Cancer tissue may also be cultured using the methods and bioreactorculture system of the disclosure. For example, adenocarcinoma cells canbe obtained by separating the adenocarcinoma cells from stromal cells bymincing tumor cells in HBSS, incubating the cells in 0.27% trypsin for24 hours at 37° C. and further incubating suspended cells in DMEMcomplete medium on a plastic petri dish for 12 hours at 37° C. Stromalcells selectively adhered to the plastic dishes.

In addition, combinations of cells include, without limitation: (a)human hepatocytes (e.g., primary hepatocytes) and fibroblasts (e.g.,normal or transformed fibroblasts, such as NIH 3T3-J2 cells); (b)hepatocytes and at least one other cell type, particularly liver cells,such as Kupffer cells, Ito cells, endothelial cells, and biliary ductalcells; and (c) stem cells (e.g., liver progenitor cells, oval cells,hematopoietic stem cells, embryonic stem cells, and the like) and humanhepatocytes and/or other liver cells and a stromal cell (e.g., afibroblast). Other combination of hepatocytes, liver cells, and liverprecursor cells.

In another aspect, certain cell types have intrinsic attachmentcapabilities, thus eliminating a need for the addition of serum orexogenous attachment factors. Some cell types will attach toelectrically charged cell culture substrates (e.g., electrically chargedspots or microwells) and will adhere to the substrate via cell surfaceproteins and by secretion of extracellular matrix molecules. Fibroblastsare an example of one cell type that will attach to cell culturesubstrates under these conditions.

In one embodiment, user defined ECM protein mixtures are deposited asdiscrete “spots” into each well of a 96-well, or similar, culture plate(FIG. 2B). Cells can then be cultured in the wells, and adhere to theprotein spotted domains preferentially under appropriate conditions,creating a cellular array. If desired, a second cell type can then beadded to the culture regions unoccupied by the first cell type, thusallowing for co-culture of two cells types. Alternatively, the culturesurface surrounding the protein spots can be modified such that cellscannot attach to or migrate to the regions surrounding the proteinspots. In this manner, mono-culture or co-culture involving multiplecells types and multiple ECM coatings can be tested in a single 96-wellplate. Assays can be performed using standard techniques which includethe use of absorbance and fluorescence plate readers.

The various techniques, methods, and aspects of the invention describedabove can be implemented in part or in whole using computer-basedsystems and methods. Particularly, the regulation of spot size andlocation on a substrate can be regulated by a computer system operablyconnected to a spotting device (e.g., a DNA spotting device).Additionally, computer-based systems and methods can be used to augmentor enhance the functionality described above, increase the speed atwhich the functions can be performed, and provide additional featuresand aspects as a part of or in addition to those described elsewhere inthis document. Various computer-based systems, methods andimplementations in accordance with the above-described technology arepresented below.

A processor-based system can include a main memory, preferably randomaccess memory (RAM), and can also include a secondary memory. Thesecondary memory can include, for example, a hard disk drive and/or aremovable storage drive, representing a floppy disk drive, a magnetictape drive, an optical disk drive, etc. The removable storage drivereads from and/or writes to a removable storage medium. Removablestorage medium refers to a floppy disk, magnetic tape, optical disk, andthe like, which is read by and written to by a removable storage drive.As will be appreciated, the removable storage medium can comprisecomputer software and/or data.

In alternative embodiments, the secondary memory may include othersimilar means for allowing computer programs or other instructions to beloaded into a computer system. Such means can include, for example, aremovable storage unit and an interface. Examples of such can include aprogram cartridge and cartridge interface (such as the found in videogame devices), a movable memory chip (such as an EPROM or PROM) andassociated socket, and other removable storage units and interfaces,which allow software and data to be transferred from the removablestorage unit to the computer system.

The computer system can also include a communications interface.Communications interfaces allow software and data to be transferredbetween computer system and external devices. Examples of communicationsinterfaces can include a modem, a network interface (such as, forexample, an Ethernet card), a communications port, a PCMCIA slot andcard, and the like. Software and data transferred via a communicationsinterface are in the form of signals, which can be electronic,electromagnetic, optical or other signals capable of being received by acommunications interface (e.g., information from flow sensors in amicrofluidic channel or sensors associated with a substrates X-Ylocation on a stage). These signals are provided to communicationsinterface via a channel capable of carrying signals and can beimplemented using a wireless medium, wire or cable, fiber optics orother communications medium. Some examples of a channel can include aphone line, a cellular phone link, an RF link, a network interface, andother communications channels. In this document, the terms “computerprogram medium” and “computer usable medium” are used to refer generallyto media such as a removable storage device, a disk capable ofinstallation in a disk drive, and signals on a channel. These computerprogram products are means for providing software or programinstructions to a computer system. In particular, the disclosureincludes instructions on a computer readable medium for calculating theproper O₂ concentrations to be delivered to a bioreactor systemcomprising particular dimensions and cell types.

Computer programs (also called computer control logic) are stored inmain memory and/or secondary memory. Computer programs can also bereceived via a communications interface. Such computer programs, whenexecuted, enable the computer system to perform the features of thedisclosure including the regulation of the location, size and content ofa microspot or microwell on a substrate.

In an embodiment where the elements are implemented using software, thesoftware may be stored in, or transmitted via, a computer programproduct and loaded into a computer system using a removable storagedrive, hard drive or communications interface. The control logic(software), when executed by the processor, causes the processor toperform the functions of the invention as described herein.

In another embodiment, the elements are implemented primarily inhardware using, for example, hardware components such as PALs,application specific integrated circuits (ASICs) or other hardwarecomponents. Implementation of a hardware state machine so as to performthe functions described herein will be apparent to person skilled in therelevant art(s). In yet another embodiment, elements are implanted usinga combination of both hardware and software.

The methods and systems of the invention have been demonstrated as setforth in more detail in the Examples. The utility of this approach, forexample, has been demonstrated by application to two different celltypes: assessing the differentiated function of mature, primary rathepatocytes by albumin immunostaining, and differentiation of murineembryonic stem (ES) cells along the hepatic lineage as assessed by aβ-galactosidase reporter on an early liver-specific gene, Ankrd17(gtar).

In one aspect, fabrication of a microarray for cell culture (e.g., anECM microarray) was obtained by optimization of deposition methods,printing buffers, microarray surfaces, and cell culture conditions. Inone aspect, a contact style arrayer using pins that deposit 1-2 nL,although larger amounts can be used, of material per ˜150 μm (e.g.,90-200 μm) diameter spot—large enough to accommodate about 5-50 cells(e.g., about 20 cells)—can be used. In some instances (e.g., where ECMmaterials are used) a modified spotting buffer is used. The modifiedbuffer comprises an acidic buffer to inhibit collagen polymerization, 5mM EDTA (to prevent laminin polymerization), triton X-100 (to reducesurface tension), and glycerol (to slow evaporation and increase thevolume of material deposited). In addition, cleaning and maintenancebetween fabrication steps or runs can be performed. For example, dippingthe pins in dimethyl sulfoxide (DMSO) between wells, and routine (e.g.,daily) sonication of the pins, can assist in reducing defects duringconsecutive printing runs.

The working examples provided below are to illustrate, not limit, thedisclosure. Various parameters of the scientific methods employed inthese examples are described in detail below and provide guidance forpracticing the disclosure in general.

EXAMPLES ECM Microarray Fabrication

The microarray substrate was a custom fabricated acrylamide gel padslide, similar to the Hydrogel slide (Perkin Elmer). To summarize, glassslides were modified with 3-(trimethoxysilyl)propyl methacrylate (Sigma)to present methacrylate groups which bond the gel to the glass. A thin(˜80 μm) polyacrylamide gel pad was created by floating an untreated22×22 mm #1 coverslip on a 40 μL drop of prepolymer solution, andexposing to UV at 1.5 mW/cm² for 10 minutes (Glo-Mark Systems, Inc.).The prepolymer solution consisted of 9.5% acrylamide, 0.5% bis, and 20mg/mL 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one(Irgacure 2959, Ciba Specialty Chemicals; dissolved initially inmethanol at 200 mg/mL). After carefully removing the coverslip, theslides were soaked in dH₂O for 48 hours and dehydrated on a hotplate at40° C. The printing buffer consisted of 100 mM acetate, 5 mM EDTA, 20%glycerol, 0.25% triton X-100 adjusted to pH=5.0 to inhibit proteinpolymerization. For ECM arrays, stock solutions of rat collagen I⁸,human collagen III, mouse collagen IV, human fibronectin (BectonDickinson), and mouse laminin (Sigma) were suspended at 500 μg/mL in theprinting buffer. ECM protein solutions were then mixed in 32combinations in a 384-well plate. Eight individual spots of each proteinmixture were deposited with a 500 μm pitch on the acrylamide gel padusing a SpotArray 24 (Perkin Elmer) equipped with Stealth SMP 3.0 splitpins (Telechem). The pins were cleaned by sonication in 5% MicroCleaning Solution (Telechem) and dH₂O immediately before use. Betweeneach sample in the source plate, the pins were dipped in a 50% DMSO andwater solution, washed for 25 seconds with dH₂O, and dried. Twenty ECMmicroarrays could be produced simultaneously in this manner in one hour.A silicone well isolator (Grace Biolabs) was adhered around the gel padusing “Silicone II” sealant (General Electric) to define the cellculture area. The protein arrays with gaskets were incubated at 4° C. ina humidified environment for ˜16 hours, and rinsed in PBS before use.

Cellular Function.

Images of the 9 mm×9 mm array were acquired at 10× as a series of 154images on a Nikon inverted microscope equipped with a motorized stage(Ludl Electronic Products Ltd.). The images were montaged usingMetamorph 6.2r3 software (Universal Imaging Corp.). Hepatocyte arrayswere fixed on days 1 and 7 in 4% paraformaldehyde and stained forintracellular albumin using a rabbit anti-rat albumin antibody (Cappel)and a goat anti-rabbit IgG-Alexa 633 secondary (Molecular Probes).Arrays were mounted in Slowfade Light (Molecular Probes) and imagedusing 3 second exposures for each frame (CoolSnap HQ, Photometrics).

I114 ES cell reporter expression was assessed at days 3 and 5. Cellarrays were fixed for 20 minutes in 0.5% glutaraldehyde and stained in0.1% X-gal in a Tris buffer (pH 7.5) overnight at 37° C. Montaged imagesof each array were acquired in bright field. β-galactosidase image areawas quantified by color thresholding using Metamorph software.

Indirect Immunofluorescence.

Five identically fabricated ECM microarrays were blocked using a 10%goat serum, 1% BSA solution in PBS. Indirect immunofluorescence wasconducted using the following primary antibodies (all raised inrabbits), and an Alexafluor 633 goat anti-rabbit secondary (1:50dilution, Molecular Probes): anti-rat collagen I (Chemicon), anti-mouselaminin (Chemicon), anti-human collagen III (Biodesign), anti-mousecollagen IV (Biodesign), and anti-human fibronectin (Sigma). Fluorescentimages were acquired using a ScanArray 4000 confocal laser scanner (GSILumonics).

Cell Culture.

ECM microarray slides with silicone gaskets were placed in sterile P-100culture dishes. The gasket area was filled with 300 μL of dH₂O. Proteinarrays were sterilized by exposure to UV in a laminar flow hood for 15minutes, followed by rinsing in sterile culture media. ECM microarrayslides with silicone gaskets were placed in sterile P-100 culturedishes. Hepatocytes were isolated from 2- to 3-month-old adult femaleLewis rats (Charles River Laboratories, Wilmington, Mass.) weighing180-200 grams, by a modified procedure of Seglen. Detailed proceduresfor isolation and purification of hepatocytes were described by Dunn etal. Routinely, 200-300 million cells were isolated with viabilitybetween 85 and 95%, as judged by trypan blue exclusion. Nonparenchymalcells, as judged by their size (<10 μm in diameter) and morphology(nonpolygonal or stellate), were less than 1%. Culture medium wasDulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10%fetal bovine serum (FBS, Sigma, St. Louis, Mo.), 0.5 U/mL insulin, 7ng/mL glucagon, 20 ng/mL epidermal growth factor, 7.5 g/mLhydrocortisone, 100 U/mL penicillin, and 100 g/mL streptomycin.Hepatocytes were suspended at 10⁶ cells/mL in culture media. The cellsuspension was dispensed onto the ECM microarray in the gasket region(0.3 mL), and incubated for ˜2 hours to allow for cell attachment(shaking the plates every 15 minutes to redistribute the cells). Thearrays were then gently aspirated to remove unattached cells and freshculture media was added to the silicone wells. Culture media was changeddaily. The I114 mouse embryonic stem cell line containing a gene-trapwas propagated in an undifferentiated state on gelatinized flasks inculture media containing 1000 U/mL leukemia inhibitory factor (LIF,Chemicon). ES media consisted of GMEM/BHK21 (Gibco) supplemented with15% FBS (Hyclone, screened for ES culture), non-essential amino acids,and 10⁻⁴ M 2-mercaptoethanol (Sigma). Cells were passaged at ˜80%confluence (approximately every two days). ECM microarrays containing450,000 cells were cultured for 6-10 hours (without shaking) to allowfor cell attachment before rinsing with fresh media. ES cell arrays werecultured for up to six days with either 1000 U/mL LIF or 10⁻⁶ Mall-trans-retinoic acid (Sigma).

Cellular Function.

Hepatocyte cell viability was assessed on day 1 and day 5 after platingusing a live/dead assay (Calcein AM and ethidium homodimer-1, MolecularProbes Inc). Cell arrays were imaged live, and then fixed in 4%paraformaldehyde and mounted in SlowFade Light w/DAPI (Molecular ProbesInc.) for high resolution microscopy. I114 ES cell alkaline phosphataseactivity was assessed by a substrate kit IV (SK-5400 VectorLaboratories). F-Actin was visualized using Phalloidin-TRITC (Sigma).Confocal volume images were acquired at 20× and 40× on a BioRad MRC1000, and digitally sectioned in the x-z plane using Metamorph 6.2r3software (Universal Imaging Corp.).

Statistics and Data Analysis.

Unless otherwise specified, all data are reported as average value±standard deviation. We assessed intracellular albumin content ofhepatocytes using Metamorph image analysis software. To quantify spotintensity, we calculated the average pixel value within a masked region(each ECM microarray contained 256 spots). After a log transformation,the data appeared to be normally distributed with approximately equalvariance. For each matrix mixture, the eight replicate log spotintensities were used to calculate the average signal and standard errorfor the condition. Two day 1-, and four day 7-arrays were quantified inthis manner. The mean signal for each array was adjusted to an arbitrarycommon value among arrays. All normalized day 7 data were analyzed as a25 full factorial design with 4 blocks (one for each microarray) usingMinitab statistical software (Minitab, State College, Pa.). Maineffects, 2-factor, 3-factor, and 4-factor interactions, along with thestatistical significance of each of these properties, were calculatedusing standard factorial analysis formulae. The residuals were normallydistributed with approximately equal variance around a mean value ofzero. β-galactosidase analysis of ES cultures was performed similarlyusing data from 4 day-3 arrays.

ECM Microarray Fabrication and Characterization.

Some of the design criteria were: 1) minimal protein usage; 2)reproducible fabrication; 3) low protein carryover; 4) non-adhesivemicroarray surface for cells; 5) compatibility with diverse cell types;6) maintain cell patterns for ˜1 week in serum media; and 7) compatiblewith conventional microscopy. A contact deposition type microarrayerover a piezo dispenser fit these criteria because it can function withas little as 3 μL of source material. Several commercially microarraysurfaces were tested for their ability to confine hepatocytes tocollagen I islands (a model ECM protein) for 48 hours of culture with10% serum. Hydrogel (Perkin Elmer), CodeLink (Amersham), and acrylamideslides maintained spatially confined cellular islands for ˜48 hours(FIG. 9A-C). A wide range of spotting solution concentrations werepermissive for cell attachment.

Protein immobilization was verified and assessed to determine whethersubstantial carryover occurred during the fabrication process. Spatiallocalization of ECM proteins was determined using antigenic recognition.In each case, a high level of fluorescence corresponded to the expectedspatial distribution of the five matrix proteins used. Collagen IVstaining showed dim fluorescence in some laminin regions, likelycorresponding to a reported 4% cross reactivity of the antibody asassessed by radio-immunoassay (Biodesign International). Alternatingtest solutions of FITC-collagen I and “buffer only” were also spottedwith the same pin. No signal was detected in “buffer only” rows using aconfocal laser scanner. Thus no protein carryover contamination wasdetected using two separate techniques.

Primary rat hepatocytes adhered to protein spotted regions, and did notattach to the acrylamide gel regions lacking ECM proteins. The cellpatterning was robust over a large surface area (9 mm×9 mm), yielding auniform array of near confluent cellular islands with a diameter of 150μm±5.8 μm (N=15). Cells were confined to the spotted regions for aperiod of at least 7 days, after which the most common mode of failurewas gel detachment from the slide. Phase contrast images of the arrayshowed compact cells with polygonal morphology, distinct nuclei, andbright intercellular borders consistent with primary hepatocytes. Thecell viability (assayed with Calcein Am/Ethidium homodimer-1) at 24hours and 5 days after plating showed predominantly live cells (˜95%)with intact membranes that excluded ethidium homodimer-1 nuclearstaining.

Effect of ECM Composition on Hepatocytes and ES Cells.

In order to probe primary rat hepatocytes for the effects of ECMcomposition, cell arrays were stained immunofluorescently forintracellular albumin (a marker of liver-specific function) and analyzedat days 1 and 7. All islands exhibited similar fluorescent intensity onday 1, with an average of 6.41 log fluorescent units ±0.166 (two arraysmeasured). In contrast, day 7 arrays showed notable differences influorescent intensity that appeared to be dependent on the initialunderlying matrix composition. Quadrants 2 and 3 of the array havecollagen IV in all spots, and appeared qualitatively and quantitativelyto be brighter than quadrants 1 and 4 (FIG. 7A-B). Approximately half ofthe analyzed mixtures had an albumin signal on day 7 that was greaterthan the average of day 1 cultures (FIG. 7C). Of note, the 15 highestalbumin signals are associated with underlying matrices containingcollagen IV. In a separate experiment, hepatocytes cultured on seriallydiluted collagen IV (ranging from 31.2 μg/mL to 500 μg/mL) for five daysshowed no significant differences in intracellular albumin signal(P=0.05 for all pairs using one-way ANOVA with a multiple comparisonTukey post-hoc test, GraphPad Prizm). Taken together, the data suggestedthat the differences in liver-specific function were not simply due to adifference in collagen IV concentration but rather an interactive effectwith other ECM molecules.

Factorial analysis methods were applied to analyze all available day 7data (4 arrays=1024 data points) for main effects, 2-, 3-, and 4-factorinteractions, in addition to the statistical significance of each effect(FIG. 7D). The analysis revealed that collagen IV had the largestoverall effect on albumin signal. Among the other main effects,fibronectin also had a positive effect, though to a lesser extent thancollagen IV. Laminin and collagen III were found to negatively impactalbumin signal. In agreement with these findings, it has been previouslyreported that secreted albumin from primary rat hepatocytes is highestwhen cultured on collagen IV, and decreases when cultured on fibronectinand laminin respectively. Interestingly, a number of 2-, 3-, and4-factor interactions were also identified as statistically significant(P=0.05). The interaction of collagen I with laminin, and collagen IIIwith laminin both had positive effects. However, each of thesecomponents individually exhibited a negative effect, suggesting anon-additive interaction. Similarly, the interaction of collagen IV withfibronectin showed a negative effect, whereas individually thesecomponents displayed positive effects.

To investigate the feasibility of using matrix arrays to study stem celldifferentiation, murine embryonic stem I114 cells were cultured for upto 6 days in the presence of 1000 U/mL leukemia inhibitory factor (LIF)or 10⁻⁶ M all-trans-retinoic acid (RA). Day 1 cultures stained uniformlypositive for alkaline phosphatase (FIG. 8A). Cells cultured with LIF forthree days (FIG. 8B) grew as 3-dimensional clusters that werereminiscent of embryoid bodies (EB's) with an average diameter 224 μm±12μm (N=14). Confocal sectioning (FIG. 8B inset) indicated that islandswere ˜77 μm in thickness (likely multi-layered). When cultured with RA,the cells grew as a relatively thin sheet of thickness ˜25 μm (FIG. 8Cinset). Notably, several matrix conditions in day 3 and 5 RA-inducedcultures elicited a substantial increase of β-galactosidase reporteractivity when stained with X-gal. For example, collagen I+collagenIII+laminin+fibronectin collectively induced noticeably more reporterexpression in all replicate islands (FIG. 8D, top left images) thancells cultured on collagen III+laminin (FIG. 8D, bottom left images).Quantitative image analysis of “blue” thresholded area in day 3RA-treated arrays illuminated further trends (FIG. 8D, bar graph). Nineof the 10 highest signals were recorded from cells on matrices thatcontained collagen I, and 4 of the top 5 signals came from ECMconditions with both collagen I and fibronectin. The lowest 11 signalswere detected on matrices that lacked fibronectin, and matrices whichlacked both collagen I and fibronectin produced the lowest 7β-galactosidase signals. A 25 full factorial analysis on data from 4arrays also indicated that fibronectin and collagen I had strongpositive effects on β-galactosidase reporter expression (FIG. 8E).Again, a number of potentially counter-intuitive interaction effectswere identified (e.g. collagen I+collagen IV, and collagenI+fibronectin).

Although a number of embodiments and features have been described above,it will be understood by those skilled in the art that modifications andvariations of the described embodiments and features may be made withoutdeparting from the teachings of the disclosure or the scope of theinvention as defined by the appended claims.

1. A cell culture substrate, comprising a plurality of microspot islandsor microwells in an array, wherein the microspot islands or microwellscomprise an insoluble factor or an insoluble and soluble factor, whereinthe insoluble factor promotes cellular adhesion.
 2. The cell culturesubstrate of claim 1, wherein the substrate comprises a polymerizedbiopolymer.
 3. The cell culture substrate of claim 2, wherein thebiopolymer is a hydrogel or a relatively homogeneous slab of biopolymer.4. (canceled)
 5. (canceled)
 6. The cell culture substrate of claim 1,further comprising a plurality of microfluidic channels connecting oneor more microspot islands or microwells to a fluid flow.
 7. A method ofmaking a microarray, comprising: spotting a plurality of locations on asubstrate with an adherence material.
 8. The method of claim 7, whereinthe adherence material is an insoluble adherence material or anextracellular matrix protein.
 9. (canceled)
 10. The method of claim 7,wherein the substrate is layered with a hydrogel and wherein thehydrogel is etched at each of the plurality of locations to form amicrowell.
 11. (canceled)
 12. The method of claim 7, wherein thesubstrate further comprises fluid flow channels fluidly connecting eachlocation with a fluid flow.
 13. The method of claim 7, furthercomprising providing one or more cell types that adhere to each locationcomprising the adherence material. 14-17. (canceled)
 18. The method ofclaim 10, wherein the hydrogel is a polyacrylamide gel.
 19. The methodof claim 7, wherein the spotting is performed by a spotting device. 20.A method of making a culture substrate, comprising: spotting a materialon the substrate using a device capable of spotting from about 1 toabout 1000 nanoliters of material to generate an island of material. 21.The method of claim 20, wherein the device is a DNA spotting device. 22.The method of claim 20, wherein the material comprises an insolublefactor or a printing buffer.
 23. (canceled)
 24. The method of claim 7,wherein the printing buffer comprises 100 mM acetate, 4 mM EDTA, 20%glycerol and 0.25% Triton X-100 at pH 5.0. 25-28. (canceled)
 29. Aculture system comprising the microarray of claim 7 and one or morecell-types.
 30. An assay system comprising: contacting a microarray ofclaim 7 with one or more cell-types and measuring an activity selectedfrom gene expression, cell function, metabolic activity, morphology, anda combination thereof.
 31. The assay system of claim 30, wherein thesystem further comprising contacting the cells with a test agent oncethe cells are in stable culture on the substrate.
 32. The assay systemof claim 30, wherein a factor is present in a material of an island onthe substrate.
 33. The method of claim 7, further comprising providingone or more soluble or insoluble material/biological material thatadhere to each location comprising the adherence material.